Bifunctional compounds that permit the linkage of two or more molecules have been described. For example, bifunctional compounds for linking cytotoxic reagents to molecules for targeting cell populations are known. The bifunctional compounds must be capable of carrying and releasing these types of cytotoxic reagents in vivo, for example to provide sufficient, i.e. therapeutic, levels of the reagents in vivo, without damaging the activity of the targeting molecules. For certain applications, the formation of a conjugate containing a pH sensitive linkage between the reagent and targeting molecules of the conjugate providing release of the cytotoxic reagent in certain ranges of pH, is desirable.
Particularly useful reagents for treatment of cancers are the anthracyclines. Anthracyclines are antibiotic compounds that exhibit cytotoxic activity. Studies have indicated that anthracyclines may operate to kill cells by a number of different mechanisms including: 1) intercalation of the drug molecules into the DNA of a cell thereby inhibiting DNA-dependent nucleic acid synthesis; 2) production by the drug of free radicals which then react with cellular macromolecules to cause damage to the cells, or 3) interactions of the drug molecules with the cell membrane (Peterson et al., "Transport And Storage Of Anthracyclines In Experimental Systems And Human Leukemia", in Anthracycline Antibiotics In Cancer Therapy, Muggia et al. (Eds.), p. 132 (Martinus Nijhoff Publishers (1982); and Bachur, "Free Radical Damage", id. at pp. 97-102)). Because of their cytotoxic potential, anthracyclines have been used in the treatment of numerous cancers such as leukemia, breast carcinoma, lung carcinoma, ovarian adenocarcinoma, and sarcomas (Wiernik, "Current Status Of Adriamycin And Daunomycin In Cancer Treatment", in Anthracyclines: Current Status And New Developments, Crooke et al. (Eds.), pp. 273-94 (Academic Press 1980)). Commonly used anthracyclines include adriamycin (ADM also known as doxorubicin) and daunomycin (DAU also known as daunorubicin).
Although these compounds may be useful in the treatment of neoplasms and other disease states wherein a target cell population is sought to be reduced or eliminated, their therapeutic efficacy is often limited by the dose-dependent toxicity associated with their administration. For example, in the treatment of tumors, typical adverse side effects of these compounds include myelosuppression and cardiotoxicity (Crooke, "Goals For Anthracycline Analog Development At Bristol Laboratories", Anthracyclines: Current Status And New Developments, supra, at p. 11). Attempts have therefore been made in the treatment of tumors to improve the therapeutic effects of these compounds by linking the anthracycline to antibodies directed against tumor-associated antigens to form immunoconjugates for selective delivery of the drugs to tumor cells. (Hermentin and Seiler, "Investigations with monoclonal antibody drug (anthracycline) conjugates", Behring Insti. Mitl. 82:197-215 (1988)). In this way, the drug can be delivered or "targeted" to the tumor site and its toxic side effects on normal cells in the body may be diminished. Immunoconjugates comprised of the anthracyclines ADM or DAU linked to polyclonal or monoclonal antibodies to tumor-associated antigens are known in the art (e.g. Gallego et al., "Preparation Of Four Daunomucin-Monoclonal Antibody 791IT/36 Conjugates With Anti-Tumor Activity", Int. J. Cancer 33: 737-44 (1984); and Arnon et al., "In Vitro And In Vitro Efficacy Of Conjugates Of Daunomycin With Anti-Tumor Antibodies", Immunological Rev. 62:5-27 (1982)).
The most frequently used approaches for the attachment of an anthracycline to an antibody have utilized a linkage at the amino sugar moiety of the anthracycline. For example, the amino sugar has been oxidized by sodium periodate treatment and directly attached to lysine residues on the antibody via Schiff base formation (Hurwitz et al., "The Covalent Binding Of Daunomycin And Adriamycin To Antibodies, With Retention of Both Drug And Antibody Activities", Cancer Res. 35:1175-1181 (1975)). Alternatively, anthracyclines have been linked to antibodies through carbodiimide-mediated linkage of the amino group of the anthracycline to carboxyl groups on the antibody (Hurwitz et al., supra) or an aminoalkyl group (Hurwitz et al., "The Effect in vivo of Chemotherapeutic drug-antibody conjugates in two murine experimental tumor systems" Int. J. Cancer 21:747-755 (1978)). These linkages are not easily hydrolyzed and make it difficult to control the release of the anthracycline. Anthracyclines have also been linked to antibodies by cross-linking the amino sugar of the drug and amino groups on the antibody with glutaraldehyde (Belles-Isles et al., "In Vitro Activity of Daunomycin-Anti-AlphaFetoprotein Conjugate On Mouse Hepatoma Cells", Br. J. Cancer 41, pp. 841-42 (1980)). However, studies with immunoconjugates in which the amino sugar portion of the anthracycline molecule was modified by linkage to the antibody indicate a loss of cytotoxic activity of the conjugated drug (Arnon et al., supra, at pp. 7-8). In addition, studies of anthracycline analogs indicate that modifications of anthracyclines at their amino sugars result in a decrease in the cytotoxic activity of the drug analog relative to the parent drug (Yamamoto et al., "Antitumor Activity of Some Derivatives of Daunomycin At The Amino And Methyl Ketone Functions", J. Med. Chem. 15:872-75 (1972)).
Still other immunoconjugates have been prepared wherein the anthracycline DAU has been linked directly to an antibody at the carbon-14 (C-14) position of the drug. However, the selective cytotoxic activity of these immunoconjugates toward tumor cells was not easily reproducible and was revealed consistently only at a concentration of 20 .mu.g/ml (Gallego et al., supra).
Japanese patent application 274658 discloses the conjugation of an anthracycline to an antibody via a C-13 acylhydrazone linkage. This conjugation was accomplished using methods that involve derivatization of the antibody and subsequent reaction of that derivative with anthracycline. These methods are not favored because derivatization of the antibody involves undesirable non-specific reactions and yields very low anthracycline:antibody ratios. According to the first method, the antibody was treated with carbodiimide in the presence of hydrazine to yield a hydrazide antibody derivative which was then reacted with the anthracycline such that the anthracycline was linked directly to the antibody structure. The resulting immunoconjugates, however, are prone to aggregation of the antibody molecules. Furthermore, because this method requires carboxylic groups which may be limited in number, these immunoconjugates have low anthracycline:antibody ratios (approximately 1.1-1.3). The second method involves reacting the antibody with succinic anhydride to yield a hemi-succinate derivative of the antibody. This derivative was next reacted with hydrazine to yield an antibody hydrazide derivative which was then reacted with the anthracycline, daunomycin. This second approach is flawed in that the reaction of the antibody derivative with hydrazine is non-specific, leading to the production of a mixture of different antibody derivatives in addition to the desired hydrazide derivative. Thus, as indicated in the 274658 application, the molar ratio of anthracycline to antibody was very low (approximately 1, see Japanese application, page 264, column 1). See also, European patent application, Publication No. 294294, which discloses the conjugation of a C-13 hydrazone derivative of an anthracycline to the carbohydrate moiety of an antibody.
Other anthracycline hydrazones are disclosed in Tong et al., J. Med. Chem., 21:732-37 (1978); Smith et al., J. Med. Chem.. 21:280-83 (1978); and Brownlee et al., J. Chem. Soc., pp. 659-61 (1986). See also U.S. Pat. No. 4,112,217, which discloses bis-hydrazones of DAU and ADM. In other studies, anthracyclines have been linked to high molecular weight carriers, such as dextran or polyglutamic acid, in order to potentiate the cytotoxic activity and reduce the toxicity of the drug (Arnon et al., supra, at p. 5 and Hurwitz et al., "Soluble Macromolecules As Carriers For Daunorubicin", J. Appl. Biochem. 2, pp. 25-35 (1980)). These carrier-linked anthracyclines have also been covalently bound to antibodies directed against tumor-associated antigens to form immunoconjugates for targeting of the cytotoxic drug specifically to tumor cells. For example, ADM has been linked to such an "anti-tumor" antibody via a carboxy-methyl-dextran hydrazide bridge wherein the ADM molecule was linked to a hydrazine derivative of carboxymethyl dextran at the C-13 carbonyl of the ADM to form a hydrazone. The antibody was then linked to the dextran hydrazide derivative with glutaraldehyde to form an adriamycin-dexantibody conjugate (Arnon et al., "Monoclonal Antibodies As Carriers For Immunotargeting Of Drugs", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (Eds.), pp. 365-83 (1985) and Hurwitz et al., "A Conjugate 0f Adriamycin And Monoclonal Antibodies To Thy-1 Antigen Inhibits A Human Neuroblastoma Cells In Vitro", Ann. N.Y. Acad. Sci. 417, pp. 125-36 (1983)).
However, the use of carriers entails certain disadvantages. For example, carrier-containing immunoconjugates are quite large in size and are removed rapidly by the reticuloendothelial system in vivo (Dillman et al., "Preclinical Trials With Combinations And Conjugates Of T101 Monoclonal Antibody And Doxorubicin", Cancer Res. 46:4886-91 (1986)). This rapid removal of the carrier-containing immunoconjugates may not be advantageous for therapy because the conjugated drug may never reach its intended site of action, i.e., the target group of cells to be killed. In addition, the presence of the high molecular weight carrier may negatively affect the stability of the immunoconjugate and has been shown to reduce the binding activity of the antibody of the conjugate (Embleton et al., "Antibody Targeting Of Anti-Cancer Agents", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (Eds.), pp. 323-24 (1985)). Furthermore, in studies with tumor cells, there is no evidence that high molecular weight carrier-containing immunoconjugates are able to localize to the tumor cells in vivo. (Compare Ford et al., "Localization And Toxicity Study Of A Vindesine-Anti-CEA Conjugate In Patients With Advanced Cancer" , Br. J. Cancer 47:35-42 (1983), which demonstrates localization of directly-conjugated drug-antibody conjugates to tumor cells in vivo).
Thus, the conjugation of anthracyclines to antibodies by the use of specific linkages and carriers has been disclosed. As outlined above, the use of these immunoconjugates entails distinct disadvantages depending upon the specific linkage or carrier used.
Certain ligand-toxin conjugates have also been disclosed. U.S. Pat. No. 4,545,985, issued to Pastan, discloses an exotoxin conjugate wherein Pseudomonas exotoxin (PE) is linked to EGF in a ratio of 1:2 for use against cells having large numbers of EGF receptors. EGF-ricin A and EGF-diphtheria toxin conjugates have also been made; Cawley et al., "Epidermal Growth Factor-Toxin A Chain Conjugates:" EGF-Ricin A Is A Potent Toxin While EGF-Diphtheria Fragment A Is Nontoxic", Cell 22:563-70 (1980) and Shimizu et al., "A Cytotoxic Epidermal Growth Factor Cross-Linked To Diphtheria Toxin A-Fragment", FEBS Letters 118 (No.2):274-78 (1980)). Furthermore, Pseudomonas exotoxin fusion proteins have been prepared using proteins, polypeptides and growth factors such as TGF-.alpha., IL-2, IL-6 and CD4 (Pastan et al., "Novel Cytotoxic Agents Created By The Fusion Of Growth Factor And Toxin Genes", Fourth Internatl. Conference On Monoclonal Antibody Immunoconjugates For Cancer, p. 36 (Mar. 30-Apr. 1, 1989); Lorberboum et al., Proc. Natl. Acad. Sci. USA, 85:1922-26 (1988); Chaudhary et al., Proc. Natl. Acad. Sci. USA, 84:4538-42 (1987); Siegall et al., Proc. Natl. Acad. Sci. USA, 85:9738-42 (1988); and Chaudhary et al., Nature, 335:369-72 (1988)). A diphtheria toxin-.alpha.-melanocyte stimulating hormone fusion protein has been made (Murphy et al., "Genetic Construction, Expression And Melanoma-Selective Cytotoxicity Of A Diphtheria Toxin-Related .alpha.-Melanocyte-Stimulating Hormone Fusion Protein", Proc. Natl. Acad. Sci. USA, 83:8258-62 (1986), and U.S. Pat. No. 4,675,382, issued to Murphy). Ligand conjugates comprising protein toxins, however, may prove to be immunogenic in xenogeneic hosts.
In addition, anthracyclines such as ADM or DAU have been chemically linked to certain protein or polypeptide ligands such as transferrin (United Kingdom patent application, GB 2116979 A) and melanotropin (Varga et al., "Melanotropin-Daunomycin Conjugate Shows Receptor-Mediated Cytotoxicity For Cultured Murine Melanoma Cells", Nature 267: 56-58 (1977)). PCT patent application WO 88/00837 describes EGF linked via a polymeric carrier to a cytotoxic substance such as DAU and U.S. Pat. Nos. 4,522,750 and 4,590,001 describe transferrin linked to vinca alkaloid and platinum, respectively.
The cytotoxic drug to be used in the immunoconjugate should be released via a conditional release mechanism, i.e. the cytotoxic drug should be released at a specific site rather than by a gradual, nonspecific site hydrolysis. It has been proposed that particular immunoconjugates are translocated to lysosomes (deDuve, "Lysosomes Revisited", Eur. J. Biochem. 137:391-397 (1983)), which are slightly acidic (pH 5.0 to 5.5) (Poznansky and Juliano, "Biological Approaches to the Controlled Delivery of Drugs: A Critical Review", Pharmacol. Rev. 36:277-336 (1984)). The use of acidic conditions to release conjugated drug has been reported in the development of cis-aconityl linkers to ADM (Shen and Reiser, "Cis-Aconityl Spacer Between Daunomycin and Macromolecular Carriers: A Model of pH-sensitive Linkage Releasing Drug From a Lysosomotrophic Conjugate", Biochem. Biophys. Res. Commun. 102:1048-1054 (1981) and Yang and Reisfeld, "Doxorubicin Conjugates With a Monoclonal Antibody Directed to a Human Melanoma-Associated Proteoglycan Suppresses the Growth of Established Tumor Xenografts in Nude Mice", Proc. Natl. Acad. Sci. 85:1189-1193 (1988)), and ketal linkers to diphtheria toxin (Srinivasachar and Neville, "New Protein Cross-Linking Reagents That Are Cleaved by Mild Acid", Biochemistry 28:2501-2509 (1989)).
Greenfield et al. have recently described the formation of acid-sensitive immunoconjugates containing the acylhydrazine compound, 3-(2-pyridyldithio)proprionyl hydrazide conjugated via an acylhydrazone bond to the 13-keto position of the anthracycline molecule, and conjugation of this anthracycline derivative to an antibody or ligand molecule (Greenfield et al., European Patent Application No. 328,147, published Aug. 16, 1989).
It would be useful to provide additional bifunctional compounds that are structured to provide acid-sensitive linkage between molecules, including targeting and reagent molecules for use in therapy in vivo.